Optogenetic control of endothelial cells

ABSTRACT

The invention features methods for regulating vascular properties by controlling the membrane properties of endothelial cells using optogenetics and light. The invention features methods to transport therapeutics across the vascular barrier into tissues such as the brain and the lung, with high spatial and temporal precision, and for controlling vascular properties such as vascular tone, arterial diameter, and vascular growth.

STATEMENT AS TO FEDERALLY FUNDED RESEARCH

This invention was made with government support under Grant No. F32NS078895, awarded by the National Institutes of Health. The governmenthas certain rights in this invention.

BACKGROUND OF THE INVENTION

The invention features methods for regulating vascular properties bycontrolling endothelial cell behavior using optogenetic reagents andlight, and uses thereof. In certain embodiments, the invention featuresmethods for regulating the permeability of the blood-brain barrier fordelivery of therapeutics to the brain and for regulating vascular toneand vascular growth.

Drugs injected via intravascular (IV) injections provide an optimalpathway for the non-invasive delivery of therapeutics to the brain. Theprimary impediment to optimal IV use is the blood-brain barrier (BBB).This barrier is crucial for normal brain function, preventing cross-talkbetween blood elements and the brain. In medical practice, the BBB is anobstacle and prevents almost all beneficial drugs from entering thebrain.

Given the importance of BBB opening for drug delivery, severalapproaches have been attempted to open or circumvent the BBB. Agentssuch as mannitol, which creates a hyper-osmotic condition, or adenosine,are non-specific in space—they open the entire BBB—and at best haveminimal open duration of tens of minutes and often compromise the BBBfor hours. A second ‘global’ approach has been drug synthesis to mimicelements that naturally pass the BBB. These ‘trojan horses’ emulatesmall molecules to use their BBB ferrying systems, or engage largemolecule receptor mediated transfer. This approach has significantpromise, but suffers from the basic challenge of spatial and temporalprecision endemic to any existing IV method, and it requires molecularengineering for each agent (alterations that could impact drugefficacy).

Currently, a single established method claims to provide non-invasivespatio-temporal precision in BBB opening—localized ultrasound pulsing ofmicro-bubbles injected IV. The mechanisms underlying this breach of theBBB are not known, but are believed to occur through mechanicalaggravation of the endothelial cell (EC) layer that is the primaryconstituent of the BBB. This method is undesirable for several reasons.First, each opening of the BBB is technically complex—magnetic resonanceimaging (MRI) is required to guide focusing of ultrasound to obtainspatial specificity. Second, the ‘transient’ opening achieved by thistechnique compromises the BBB for at least 3-5 hours. Third, the methodworks through an aphysiological mechanical BBB rupture, which isunlikely to allow multiple uses without creating chronic localreactivity or damage.

Thus there is a need for non-invasive and simple methods for theregulation of vascular permeability, especially in the BBB, with highspatial and temporal precision for delivery of therapeutics.

Several maladies result from failures in proper vascular tone, arterialdiameter or improper vascular growth (angiogenesis). Thus there is alsoa need for non-invasive methods for controlling vascular tone, arterialdiameter and vascular growth.

SUMMARY OF THE INVENTION

The invention features a method for changing the permeability ofendothelial cells. The method generally includes the steps of contactingendothelial cells expressing optogenetic reagents with light andactivating the optogenetic reagents with light, thereby changing thepermeability of the endothelial cells.

In one embodiment, the invention features a method for changing thepermeability of endothelial cells by a) infecting endothelial cells withrecombinant viruses comprising a recombinant nucleic acid encoding anoptogenetic reagent to produce infected endothelial cells; and b)contacting the infected endothelial cells with light, wherein the lightactivates the optogenetic reagents and thereby changes the permeabilityof the endothelial cells.

In another embodiment, the invention features a method to deliver atherapeutic across the blood-brain barrier by a) infecting endothelialcells with recombinant viruses comprising a recombinant nucleic acidencoding the optogenetic reagent to produce infected endothelial cells;b) introducing the therapeutic (e.g., drugs, small molecules, peptides,proteins, antibodies, nucleic acid molecules, and organic and inorganiccompounds) into the blood stream; and c) contacting the infectedendothelial cells with light, to activate the optogenetic reagents andthereby change the permeability of the endothelial cells and open theblood-brain barrier such that the therapeutic in the blood streamcrosses the blood-brain barrier.

The invention also features methods to change the permeability ofendothelial cells, which are part of a blood-brain barrier, such thatthis change in permeability results in opening or closing of the BBB.Such opening and closing of the BBB results in increased or decreaseddelivery of elements (e.g., therapeutic agents) from the endothelialcells to the brain.

The methods of the invention can be used, e.g., to regulate vasculartone, regulate arterial diameter, control blood flow to a region of atissue, control delivery of blood-borne factors, and regulate vasculargrowth in a subject.

The methods of the invention can be used, e.g., to treat, or treatprophylactically a brain disease (e.g., glioma, epilepsy, Alzheimer'sdisease, multiple sclerosis, and meningitis). The method of theinvention can also be used to treat or treat prophylactically a vasculardisease caused by failure in proper vascular tone (e.g., stroke,aneurysm, diabetes, hypertension, and cardiac disease). Alternatively,the methods of the invention can be used to treat or treatprophylactically a disease caused by abnormal vascular growth (e.g.,retinopathy, cerebrovascular epilepsy, and cancer).

Another aspect of the invention features methods to change thepermeability of endothelial cells that are part of the blood-air barrierin the lung. These methods can be used to treat a disease of the lung(e.g., lung cancer).

In the methods of the invention, the optogenetic reagents can be, forexample, ChR1, ChR2, VChR1, ChR2 C128A, ChR2 C128S, ChR2 C128T, ChD,ChEF, ChF, ChIEF, NpHR, eNpHR, Arch 3.0, Arch T 3.0, Mac 3.0,melanopsin, chimeras of these proteins, or natural or engineeredvariants thereof.

In another aspect of the invention, the optogenetic reagents areexpressed in the endothelial cells by introducing a recombinant nucleicacid encoding the optogenetic reagent into the cells or precursorsthereof. The recombinant nucleic acid can be introduced into the cellsby using any suitable approach, e.g., virus, an electroporation device,transfection, or by way of a transgenic method.

If desired, the recombinant nucleic acid can be encapsidated within arecombinant virus selected from the group consisting of recombinantadeno-associated virus (AAV), recombinant retrovirus, recombinantlentivirus, recombinant poxvirus, recombinant rabies virus, recombinantpseudo-rabies virus, and recombinant herpes simplex virus, and humanimmunodeficiency virus (HIV).

The recombinant nucleic acid encoding the optogenetic reagents can alsoencode a fiducial (e.g., a fluorescent protein such as green fluorescentprotein) marker, that when expressed identifies the cells that have beeninfected by the recombinant virus indicating that these cells willexpress the optogenetic reagent.

Any route of administration can be used to deliver the virus. Forexample, the virus may be applied using intravenous injection or appliedlocally to infect endothelial cells in specific regions.

In addition, light may be applied using a laser or a light emittingdiode, and the application of light maybe restricted to a definedspatial region of the body. The light can be delivered using a fiberoptic cable or catheter. The methods of the invention also involve lightbeing shined on a specific region of the brain for a specific period oftime, thus providing spatial and temporal control of the opening of theBBB.

Definitions

The term “optogenetic control” refers to the control of physiologicalproperties of a cell by introducing a light-activated molecular channel(e.g., channnelrhodopsin-2) into the membrane of cells by genetic means;and contacting these cells with light of a wavelength that activates themolecular channel and causes a change in the membrane properties of thecell.

By “endothelial cells” is meant cells that line the interior surface ofblood vessels.

By “expressing optogenetic reagents” is meant the production of one ormore exogenous light-activated agents that impact cell physiology, mostnotably molecular channel proteins, in a cell into which a recombinantnucleic acid molecule encoding the light-activated molecular channelprotein has been introduced by genetic means.

By “optogenetic reagents” is meant natural or engineered variants oflight-activated agents that impact cell physiology , most notablymolecular channel proteins, including elements such as channelrhodopsin,halorhodopsin, melanopsin and archaerhodopsin (e.g., ChR1, ChR2, VChR1,ChR2 C128A, ChR2 C128S, ChR2 C128T, ChD, ChEF, ChF, ChIEF, NpHR, eNpHR,Arch 3.0, Arch T 3.0, Mac 3.0). Exemplary optogenetic reagents areprovided in Table 1.

By “electroporation” is meant a method of introducing exogenous nucleicacid molecules into cells by applying an external electric field thatcauses an increase in the permeability of the cell plasma membrane anduptake of the nucleic acid molecules into the cell.

By “transfection method” is meant a method of introducing exogenousnucleic acid molecules into mammalian cells by chemically opening poresin the cell membrane (e.g., by application of calcium phosphate), toallow uptake of the exogenous nucleic acid molecules. Alternatively,transfection may also be performed by mixing a cationic lipid with theexogenous nucleic acid molecules to produce liposomes that fuse with thecell membrane and deposit the exogenous nucleic acid molecules insidecells.

By “transgenic method” is meant a method of introduction of recombinantnucleic acid molecules into the genome of the organism.

By “fiducial marker” is meant a genetically encoded protein (e.g., afluorescent protein) that enables identification of cells expressing therecombinant light-activated molecular channel.

By “defined spatial region” is meant a predetermined, specific, andconfined area of the body.

By “blood-brain barrier” is meant the membrane structures andcell-to-cell contacts that protect the brain from chemicals in theblood, while still allowing essential metabolic function. Theblood-brain barrier is composed of endothelial cells, which are packedvery tightly in brain capillaries. The blood-brain barrier includes theblood-retinal barrier.

By “opening of the blood-brain barrier” is meant an increase in thepermeability of endothelial cells in the blood-brain barrier, such thatthere is an increase in the transport of elements from the blood vessel,across the blood-brain barrier, into the brain.

By “closing of the blood-brain barrier” is meant a decrease in thepermeability of endothelial cells in the blood-brain barrier, such thatthere is a decrease in the transport of elements from the blood vessel,across the blood-brain barrier, into the brain.

By “vascular tone” is meant the state of contractile tension in bloodvessel walls.

By “arterial diameter” is meant the diameter of arteries.

By “vascular growth” is meant the creation of new blood vessels.

By “vasodilation” is meant the increase in the vascular diameter. Terms“vasodilation” and “vascular dilation” are used herein interchangeably.

By “vasoconstriction” is meant the decrease in the vascular diameter.Terms “vasoconstriction” and “vascular constriction” are used hereininterchangeably.

By “luciferase” is meant an enzyme that is capable of convertingchemical energy into light.

By “luciferin” is meant a compound that upon reaction in the presence ofa luciferase emits light.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic describing the BBB Integrated Optogenetics (BBBIO)method for precise control of endothelial cell (EC) function.Optogenetic reagents are expressed system-wide in vascular ECs. Localpresentation of light of a first color, allows opening of the BBB at aprecise location in the brain. Presentation of a second color of light,driving a different optogenetic reaction, can then be used to close theBBB, providing precise temporal control of BBB permeability.

FIG. 2A is a micrograph obtained by 2-photon imaging of a mouse brainexpressing optogenetic reagents in ECs. A brief optogenetic drive ofHalohodopsin-3.0 (Halo), a photo-activated chloride pump can open theBBB and let rhodamine-dextran enter from the blood into the brain. Threedifferent regions of interest (ROI's) have been marked forquantification of the rhodamine fluorescence that entered the brain as aresult of the opening of the Halorhodopsin channel.

FIG. 2B is a graph showing the quantification of the florescence in thethree ROIs marked in FIG. 2A. The X-axis shows the time and the Y-axisshows the fluorescence of rhodamine-dextran in each of the three ROIs.

FIG. 3A is a second example of a micrograph obtained by 2-photon imagingof a mouse brain expressing optogenetic reagents in ECs. Three trials,tens seconds in duration, of brief optogenetic drive of Halohodopsin-3.0(Halo), a photo-activated chloride pump can open the BBB and letrhodamine-dextran enter from the blood into the brain. Two differentregions of interest (ROI's) have been marked for quantification of therhodamine fluorescence that entered the brain as a result of the openingof the Halorhodopsin channel for each trial. Arrows indicate visiblerhodamine fluorescence in multiple locations outside of the BBB.

FIG. 3B is a graph showing the quantification of the florescence in thetwo ROIs marked in FIG. 3A. The X-axis shows the time for three trialsand the Y-axis shows the fluorescence of rhodamine-dextran in each ofthe two ROIs.

FIG. 4A and FIG. 4B are micrographs obtained using 2-photon microscopyof a mouse arteriole that is laterally coursing from approximately theborder of neocortical layers I and II (bar=80 microns). The arteriole inFIG. 4A is dilated following vibrissal stimulation. The arteriole inFIG. 4B is constricted following sensory drive.

FIG. 4C and FIG. 4D are graphs showing the quantification of thenormalized width of an arteriole in FIG. 4A and FIG. 4B, respectively.The X-axis shows the time, and the Y-axis shows the normalized width ofthe arteriole. The vibrissal stimulation is demarcated with a graybackground.

FIG. 5A is a micrograph obtained using 2-photon microscopy of a mousearteriole labeled with EYFP.

FIG. 5B is a graph showing the normalized width of an arteriole before,during, and after administration of amber light (560 nm). Endothelialcells of the arteriole were transduced to express Halorhodopsin 3.0. TheX-axis shows the time and the Y-axis shows the normalized width of thearteriole.

FIG. 5C is a graph showing the normalized width of an arteriole before,during, and after administration of blue light (470 nm). Endothelialcells of the arteriole were transduced to express Channelrhodopsin-2.The X-axis shows the time and the Y-axis shows the normalized width ofthe arteriole.

DETAILED DESCRIPTION OF THE INVENTION

The invention features methods for regulating vascular permeability bycontrolling endothelial cell behavior using optogenetic reagents andlight. One aspect of the invention (named BBBIO) features optogeneticmethods for precise spatial and temporal control of opening and closingof the BBB in order to deliver IV-injected therapeutics to the brain byregulating the permeability of endothelial cells, the primaryconstituent of the BBB. The invention can also be used for regulatingvascular properties in other areas of the body such as the blood-lungbarrier, also lined by endothelial cells. The method can also be usedfor regulating vascular properties such as vascular tone, arterialdiameter, and vascular growth (angiogenesis). As an example, the use ofthe methods of the invention in regulating the BBB is described below.

The Blood-Brain Barrier (BBB): a major obstacle in vascular delivery oftherapeutics for treatment of brain diseases

The vasculature provides many advantages as a non-invasive pathway fordelivering drugs to the brain. Intravascular (IV) injections reach theirtargets rapidly: vascular delivery initiated in the ascending system(e.g., in the carotid artery) reaches the brain in less than half aminute, and an injection anywhere in the system has a delivery time ofminutes. The vasculature also provides remarkable access: in theneocortex, all neural somata are within <25 microns of a blood vessel.Further, this intrinsic routing network is not subject to the unevendiffusion of injections in other body areas, or the delays andimpediments of oral delivery.

However, drug delivery to the brain is impeded by the presence of theBBB. The BBB shields the brain against potentially toxic substancespresent in the blood stream and is crucial for normal brain function,preventing cross-talk between blood elements and neural signaling andbalancing metabolic delivery.

Endothelial cells lining the blood vessels constitute the main componentof the BBB. Endothelial cells (ECs) are closely sealed by tightjunctions, possess few fenestrae and few endocytic vesicles as comparedto capillaries of other organs. The ECs are surrounded by extracellularmatrix, astrocytes, pericytes, vascular smooth muscle cells, andmicroglial cells. The close association of endothelial cells with theastrocyte foot processes and the basement membrane of capillaries areimportant for the development and maintenance of the BBB properties thatpermit tight control of blood-brain exchange.

The BBB presents passive and active resistance to brain permeability.The proximity of tight junctions (TJ) between brain ECs limitintercellular trans-laminar flow to small hydrophilic molecule. Thosemolecules that cross the EC by trans-cellular, non-receptor mediatedmeans are lipophilic (logP˜4) and low molecular weight (e.g., <400 D).However, most molecules fitting this profile do not cross the BBBbecause active efflux transporters, such as the permeabilityglycoprotein (e.g., P-gp), extrude them. Select small molecules (e.g.,amino acids) are relayed by transport proteins, and a few largemolecules by receptor-mediated trans-cytosis. Thus the BBB prevents 98%of all small-molecule therapeutics, and essentially 100% of allunmodified large-molecule therapeutics from entering the brain. Because,peptide and protein therapeutics are generally excluded from transportfrom blood to brain, owing to the negligible permeability of the braincapillary endothelial wall to these drugs, ECs represent the majorobstacle for the use of potential therapeutics against many disorders ofthe CNS.

There is extensive evidence that multiple factors under optogeneticcontrol can have a powerful impact on BBB permeability: calcium,membrane potential, chloride, and dilation. Manipulation ofextracellular calcium influx profoundly impacts EC permeability.Mechanisms include manipulation of TJ width by calcium impact oncytoskeletal proteins (e.g., regulating phosphorylation). Several linesof evidence indicate EC calcium concentration also regulates P-gpmediated drug resistance. Membrane potential changes also impactpermeability, effects likely mediated by P-gp modulation. The EC alsopossess several chloride channels, and chloride regulation has long beenbelieved to impact BBB permeability. Regulating calcium levels, chloridelevels, or membrane potential in EC should regulate EC permeability.Recent studies have also indicated that induction of vasodilation, forexample driven by local neural activity in the process of functionalhyperemia, can also enhance BBB permeability.

We have discovered an optogenetic method for controlling thepermeability of ECs in the vasculature. In one aspect of the invention,as described in FIG. 1, the method can be used for transportingIV-delivered therapeutics across the BBB into the brain. This isachieved by system-wide expression of optogenetic elements in ECs andlocal presentation of light in a defined area of the brain, thusachieving spatial control of BBB regulation. These optogenetic elementsinclude ion channels, chloride channels, and/or proton pumps, whosepermeability properties are reversibly changed upon presentation oflight of a specific wavelength. Light of multiple wavelengths can beused to change the permeability properties of cells between two states.Presentation of light of one wavelength (e.g., blue-green) may change ECpermeability and open or close the BBB. Presentation of light of adifferent wavelength (e.g., yellow or amber) can have the oppositeeffect. For example, light of one wavelength increases EC permeabilityand opens the BBB and light of another wavelength decreases ECpermeability and closes the BBB. Thus using light of two differentwavelengths, precise temporal control in the opening and closing the BBBcan be achieved. The invention provides a non-invasive and simple methodfor regulating the BBB, uses conventional drug delivery methods such asIV-injection, and is a viable long-term option where repeated openingand closing of the BBB may be required over a long course of treatment.

Optogenetic Reagents

The invention discloses the use of optogenetic reagents which includesion channels, chloride channels, and proton pumps. These includechannelrhodopsins, halorhodopsins, archaerhodopsins, or melanopsins,their natural variants, engineered chimeras or variants, and humanizedvariants as described in Table 1.

In one aspect of the invention, each of the channels described below isalso modified by addition of recombinant endoplasmic reticulum exportand trafficking signal for improved cell surface expression.

In one aspect of the invention, the optogenetic reagent is achannelrhodopsin, e.g., ChR1, ChR2, VChR1, ChR2 C128A, ChR2 C128S, ChR2C128T, ChD, ChEF, ChF, or ChIEF. Channelrhodopsins (ChRs) arelight-gated ion channels originating from microalgae. The Vitamin Aderivative retinal is linked to a lysine residue of the proteins(Retinal Schiff Base, RSB) constituting the light absorbing chromophore.They are activated by blue light. The blue light can have a wavelengthof approximately 470 nm (e.g., 440, 450, 460, 470, 480, 490 nm). Lightabsorption causes retinal isomerization around the 13-bond. Thisisomerization triggers subsequent conformational changes of the proteinand gating of the channel. Thermal relaxation of the proteins closes thechannel and the protein converts under re-isomerization of the retinalback to the dark state.

ChR2 from Chiamydomonas reinhardtii has been established as the ChRprototype for optogenetic application since it is more than 10 timesbetter expressed in most host cells than the earlier found ChR1. In oneaspect of the invention, the channelrhodopsin is vChR1 or vChR2 derivedfrom Volvox carteri. Variants of channelrhodopsins are listed below inTable 1.

In one aspect of the invention, the channelrhodopsin is a humanized ChR2with two mutations (E123T and H134R), and is called ChETA. ChETA hasfaster deactivation kinetics and faster recovery from inactivation.

In another aspect of the invention, the channelrhodopsin is a stepfunction opsin (SFO) with bi-stable excitation that is engineered by apoint mutation of ChR2, e.g., ChR2C128A, ChR2C128, and ChR2C128T. Eachof these channels is opened by presenting blue light (470 nm) and thechannels can be closed by shining a pulse of green light (542 nm). SFOsallow opening and closing the channel by shining light of differentwavelengths, thus providing precise temporal control over thepermeability of endothelial cells and of the BBB.

In another aspect of the invention, the channelrhodopsin is a stabilizedstep-function opsin (SSFO) that is engineered by making two mutations inChR2 (C128S and D156A). The SSFO channel has a more stabilizedconducting state with a time constant of nearly 30 minutes following abrief pulse of activating light. The SSFO may be closed using yellowlight (590 nm).

In another aspect of the invention, the optogenetic reagent is ahalorhodopsin, e.g., NpHR, eNpHR 2.0, and eNpHR 3.0. Halorhodopsins arelight-gated chloride channels originating from halobacteria and areactivated by yellow (or amber) light of approximately 570 nm wavelength(e.g., 540, 550, 560, 570, or 580 nm). The halorhodopsin fromNatronomonas pharaonis (NpHR) has been established as the prototypehalorhodopsin and has been used for engineering the variants eNpHR 2.0(Gradinaru et al., Brain Cell Biol., 2008, 36 (1-4): 129-139,incorporated herein by reference) and eNpHR 3.0 (Gradinaru et al., Cell,2010, 141 (1):154-165, incorporated herein by reference) described inTable 1.

In one aspect of the invention, the halorhodopsin is eNpHR 2.0 made byfusion of the FCYENEV ER export motif from a vertebrate inward rectifierpotassium channel to the C-terminus of the NpHR protein. In anotheraspect of the invention, the halorhodopsin is eNpHR 3.0 made by addingthe trafficking signal from Kir2.1 to the C terminus of the NpHRprotein.

TABLE 1 Organism Peak from which Excitation Channel channel wasWavelength Type of Acronym Name of channel isolated (nm) ChannelOpen/Close ChR1 Channelrhodopsin1 Clamydomonas 470 Ion Open by bluelight reinhardti channel (H+, Na+, K+, Ca2+) ChR2 Channelrhodopsin2Clamydomonas 470 Ion Open by blue light reinhardti channel (H+, Na+, K+,Ca2+) vChR1 Channelrhodopsin1 Volvox carteri 570 Ion Open by yellowlight channel (H+, Na+, K+, Ca2+) vChR2 Channelrhodopsin2 Volvox carteri470 Ion Open by blue light channel (H+, Na+, K+, Ca2+) ChR2H134RChannelrhodopsin2 Clamydomonas 450 Ion Open by blue light (mutant)reinhardti channel (H+, Na+, K+, Ca2+) ChR2E123T Channelrhodopsin2Clamydomonas 490 Ion Open by blue light (ChETA) (mutant) reinhardtichannel (faster deactivation) (H+, Na+, K+, Ca2+) ChD ChannelrhodopsinClamydomonas 450 Ion Open by blue light ½/hybrid reinhardti channel (H+,Na+, K+, Ca2+) ChEF Channelrhodopsin Clamydomonas 470 Ion Open by bluelight ½/hybrid reinhardti channel (H+, Na+, K+, Ca2+) ChIEFChannelrhodopsin Clamydomonas 450 Ion Open by blue light ½/hybridreinhardti channel (H+, Na+, K+, Ca2+) ChR2C128A Channelrhodopsin2Clamydomonas 470(open)/ Ion Step function (open (mutant) reinhardti542(close) channel by blue light and (H+, close by yellow Na+, K+,light) Ca2+) ChR2C128S Channelrhodopsin2 Clamydomonas 470(open)/ IonStep function (open (mutant) reinhardti 542(close) channel by blue lightand (H+, close by yellow Na+, K+, light) Ca2+) ChR2C128TChannelrhodopsin2 Clamydomonas 470(open)/ Ion Step function (open(mutant) reinhardti 542(close) channel by blue light and (H+, close byyellow Na+, K+, light) Ca2+) NpHR Halorhodopsin Natromonas 570 ChlorideOpen by yellow light pharaonis for inhibition eNpHR 2.0 engineeredNatromonas 570 Chloride Open by yellow light Halorhodopsin pharaonis forinhibition eNpHR 3.0 engineered Natromonas 570 Chloride Open by yellowlight Halorhodopsin pharaonis for inhibition Arch 3.0 ArchaerhodopsinHalorubrum yellow light Proton Open by yellow light sodomense pump forinhibition Arch T 3.0 Archaerhodopsin Halorubrum yellow light ProtonOpen by yellow light sodomense pump for inhibition Mac 3.0 Outwardlight-gated Leptosphaeria 542 Proton Open by yellow light proton pumpmaculans pump for inhibition

In another aspect of the invention, the optogenetic reagent is anarchaerhodopsin, e.g., Arch, Arch T, and Arch T 3.0. Archaerhodopsinsare light-driven proton pumps from the archaebacteria Halorubrumsodomense, e.g., Arch and are activated by yellow light. In yet anotheraspect of the invention, the optogenetic reagent is Arch T is derivedfrom the Halorubrum sp. TP009 strain and is 3.5 times more sensitivethan Arch.

In another aspect of the invention, the optogenetic reagent is Mac3.0and is an outward light-gated proton pump from Leptosphaeria maculans,also activated by yellow light.

In one aspect of the invention, a channelrhodopsin can be coexpressedwith a halorhodopsin to achieve bidirectional control of cell membranepermeability as described in Zhang et al (Zhang et al., Nature, 2007,446 (7136):633-639, incorporated herein by reference).

Genetic Means for Delivery of Optogenetic Reagents in to Cells

The optogenetic reagents used in the present invention can be expressedin endothelial cells or its precursors by delivery of recombinantnucleic acid molecules encoding these reagents into the cells. Therecombinant nucleic acid molecules are cloned into appropriateexpression vectors that contain regulatory elements necessary forexpression of the optogenetic reagents. The recombinant nucleic acidmolecules can be delivered into cells by any one or more methods knownin the art e.g., by a virus, by electroporation, by liposomes, or bytransgenic methods. These are described below.

Expression Vectors Containing Recombinant Nucleic Acid Molecule forExpressing Optogenetic Reagents in Cells

The invention features recombinant nucleic acid molecules encoding theoptogenetic reagents that are cloned into expression vectors. Theseexpression vectors, and the regulatory sequences used for expression ofoptogenetic reagents are described below.

Construction of vectors for recombinant expression of optogeneticreagents for use in the invention may be accomplished using conventionaltechniques which do not require detailed explanation to one of ordinaryskill in the art. For review, however, those of ordinary skill may wishto consult Maniatis et al., in Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory (NY 1982).

For generation of efficient expression vectors, it is necessary to haveregulatory sequences that control the expression of the optogeneticreagent. These regulatory sequences include promoter and enhancersequences and are influenced by specific cellular factors that interactwith these sequences.

Promoter and enhancer regions have been described in the art. Methodsfor maintaining and increasing expression of transgenes in quiescentcells include the use of promoters including collagen type I (1 and 2),SV40, and LTR promoters. According to one embodiment of the invention,the promoter is a constitutive promoter selected from the groupconsisting of: ubiquitin promoter, CMV promoter, JeT promoter (e.g., asdescribed in U.S. Pat. No. 6,555,674, incorporated herein by reference),SV40 promoter, Elongation Factor 1 alpha promoter (EF1-alpha), RSV,Mo-MLV-LTR. Examples of inducible/repressible promoters include: Tet-On,Tet-Off, Rapamycin-inducible promoter, and Mx1.

In another embodiment of the invention, the promoter is constitutive orinducible endothelial cell specific promoter selected from the groupconsisting of: a family of receptor tyrosine kinase genes specificallyexpressed in mammalian endothelial cells, including Tie1 and Tie2 (alsocalled Tek) (Dumont et al., Oncogene, 7: 1471-1480, 1992; Schnurch andRisau, Development, 119: 957-968, 1993), fms-like tyrosine kinase-1(FLT-1) (Nicklin et al., Hypertension, 38: 65-70, 2001), intercellularadhesion molecule 2 (ICAM-2) (Cowan et al., Transplantation, 62:155-160, 1996), VE-cadherin (VECD) (Hisatsune et al., Blood, 105:4657-4663, 2005), Endothelial cell-specific molecule 1 (ESM1) (Lassalleet al., J. Biol. Chem. 271: 20458-20464, 1996) and synthetic variantsthereof.

In addition to using viral and non-viral promoters to drive transgeneexpression, an enhancer sequence may be used to increase the level oftransgene expression. For example, in the present invention collagenenhancer sequences may be used with the collagen promoter 2 (I) toincrease transgene expression. In addition, the enhancer element foundin SV40 viruses may be used to increase transgene expression. Thisenhancer sequence consists of a 72 base pair repeat as described byGruss et al., Proc. Natl. Acad. Sci. USA, 1981, 78: 943; Benoist andChambon, Nature, 1981, 290: 304, and Fromm and Berg, J. Mol. Appl.Genetics, 1982, 1: 457, each of which is incorporated herein byreference. This repeat sequence can increase the transcription of manydifferent viral and cellular genes when it is present in series withvarious promoters (Moreau et al., Nucleic Acids Res., 1981, 9: 6047,incorporated herein by reference).

Further expression enhancing sequences include but are not limited toWoodchuck hepatitis virus post-transcriptional regulation element, WPRE,SP163, CMV enhancer, and Chicken β-globin insulator or other insulators.

Transgene expression may also be increased for long term stableexpression using cytokines to modulate promoter activity. Severalcytokines have been reported to modulate the expression of transgenefrom collagen 2 (I) and LTR promoters. For example, transforming growthfactor (TGF), interleukin (IL)-I, and interferon (INF) down regulate theexpression of transgenes driven by various promoters such as LTR. Tumornecrosis factor (TNF) and TGF 1 up regulate, and may be used to control,expression of transgenes driven by a promoter. Other cytokines that mayprove useful include basic fibroblast growth factor (bFGF) and epidermalgrowth factor (EGF).

Collagen promoter with the collagen enhancer sequence (Coll (E)) mayalso be used to increase transgene expression by suppressing further anyimmune response to the vector which may be generated in a treated brainnotwithstanding its immune-protected status. In addition,anti-inflammatory agents including steroids, for example dexamethasone,may be administered to the treated host immediately after vectorcomposition delivery and continued, preferably, until anycytokine-mediated inflammatory response subsides. An immunosuppressionagent such as cyclosporin may also be administered to reduce theproduction of interferons, which downregulates LTR promoter and Coll (E)promoter-enhancer, and reduces transgene expression.

The expression vector may further comprise sequences such as a sequencecoding for the Cre-recombinase protein, and LoxP sequences. A furtherway of ensuring temporary expression of the optogenetic reagent isthrough the use of the Cre-LoxP system which results in the excision ofpart of the inserted DNA sequence either upon administration ofCre-recombinase to the cells or by incorporating a gene coding for therecombinase into the virus construct. Incorporating a gene for therecombinase in the virus construct together with the LoxP sites and astructural gene (an optogenetic reagent in the present case) oftenresults in expression of the structural gene for a period ofapproximately five days or more.

Virus Mediated Delivery of Expression Vectors to Express OptogeneticReagents in Endothelial Cells

In one aspect of the invention, the expression vector containing therecombinant nucleic acid encoding the optogenetic reagent isencapsidated within a recombinant virus e.g., recombinantadeno-associated virus (AAV), recombinant retrovirus, recombinantlentivirus, recombinant poxvirus, recombinant rabies virus, recombinantpseudo-rabies virus, and recombinant herpes simplex virus, papovavirus,human immunodeficiency virus (HIV), and adenovirus. These viruses arethen applied to the subject (e.g., a patient) so that the endothelialcells can be infected by these viruses and theoptogenetic reagents canthen be expressed in endothelial cells.

Preferred viruses include lentiviruses and adeno-associated viruses(AAVs). Both types of viruses can integrate into the genome without celldivisions, and both types have been tested in pre-clinical animalstudies. Methods for preparation of AAVs are described in the art e.g.,in U.S. Pat. No. 5,677,158, U.S. Pat. No. 6,309,634, and U.S. Pat. No.6,683,058, each of which is incorporated herein by reference. Methodsfor preparation and in vivo administration of lentiviruses are describedin U.S. No. 20020037281 (incorporated herein by reference). Preferably,a lentivirus vector is a replication-defective lentivirus particle. Sucha lentivirus particle can be produced from a lentiviral vectorcomprising a 5′ lentiviral LTR, a tRNA binding site, a packaging signal,a promoter operably linked to a polynucleotide signal encoding saidfusion protein, an origin of second strand DNA synthesis and a 3′lentiviral LTR.

Retroviruses are most commonly used in human clinical trials, since theycarry 7-8 kb and since they have the ability to infect cells and havetheir genetic material stably integrated into the host cell with highefficiency (see, e.g., WO 95/30761; WO 95/24929, each of which isincorporated herein by reference). Oncovirinae require at least oneround of target cell proliferation for transfer and integration ofexogenous nucleic acid sequences into the patient.

For use in human patients, the retrovirus must be replication defective.This prevents further generation of infectious retroviral particles inthe target tissue. Instead the replication defective virus becomes a“captive” transgene stable incorporated into the target cell genome.Typically in replication defective vectors, the gag, env, and pol geneshave been deleted (along with most of the rest of the viral genome).Heterologous DNA (in case of the present invention, the recombinantnucleic acid molecule encoding the optogenetic reagent) is inserted inplace of the deleted viral genes. The heterologous genes may be underthe control of the endogenous heterologous promoter, anotherheterologous promoter active in the target cell, or the retroviral 5′LTR (the viral LTR is active in diverse tissues).

In one embodiment, the viruses are introduced into the body byintravascular injection. For localized targeting, virus injection froman IV catheter has already been used to achieve spatially discreteexpression (e.g., of a single chamber of the heart or localized cerebralvasculature). In cases where the desired target can be accessed bycatheterization, local transduction would then provide spatialspecificity to BBB opening. Alternatively, direct intra-cerebral virusinjection can be used to target specific vessels. While more invasivethan catheterization, this procedure is less invasive than implantationof a deep-brain stimulator and does not require maintenance of hardwarein the brain. Further, in cases where more elaborate surgery is alreadystandard—tumor removal, epilepsy surgery—local transduction could beachieved. Alternatively, direct peripheral virus injection can be usedto target specific vessels outside of the central nervous system.

Viruses encoding optogenetic reagents may be placed into apharmaceutically acceptable suspension, solution or emulsion. Suitablemediums include saline and liposomal preparations. More specifically,pharmaceutically acceptable carriers may include sterile aqueous ofnon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils.Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives may also be present such as,for example, antimicrobials, antioxidants, chelating agents, and inertgases and the like.

Non-Viral Methods for Delivery of Expression Vectors to ExpressOptogenetic Reagents in Endothelial Cells

The recombinant nucleic acid molecule encoding the optogenetic reagentmay be delivered into ECs by non-viral methods. For example, a colloidaldispersion system may be used for targeted gene delivery. Colloidaldispersion systems include macromolecule complexes, nanocapsules,microspheres, beads, and lipid-based systems including oil-in-wateremulsions, micelles, mixed micelles, and liposomes. Liposomes areartificial membrane vesicles that are useful as delivery vehicles invitro and in vivo. It has been shown that large unilamellar vesicles(LUV), which range in size from 0.2-4.0 μm, can encapsulate asubstantial percentage of an aqueous buffer containing large macromolecules. RNA, DNA and even intact virions can be encapsulated withinthe aqueous interior and be delivered to cells in a biologically activeform. For a liposome to be an efficient gene transfer vehicle, thefollowing characteristics should be present: encapsulation of theexpression vector at high efficiency while not compromising theirbiological activity; preferential and substantial binding to a targetcell in comparison to non-target cells; delivery of the aqueous contentsof the vesicle to the target cell cytoplasm at high efficiency; andaccurate and effective expression of genetic information.

The composition of the liposome is usually a combination ofphospholipids, particularly high-phase-transition-temperaturephospholipids, usually in combination with steroids, especiallycholesterol. Other phospholipids or other lipids may also be used. Thephysical characteristics of liposomes depend on pH, ionic strength, andthe presence of divalent cations. Examples of lipids useful in liposomeproduction include phosphatidyl compounds, such as phosphatidylglycerol,phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine,sphingolipids, cerebrosides, and gangliosides. Particularly useful arediacylphosphatidylglycerols, where the lipid moiety contains from 14-18carbon atoms, particularly from 16-18 carbon atoms, and is saturated.Illustrative phospholipids include egg phosphatidylcholine,dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine.

The targeting of liposomes can be classified based on anatomical andmechanistic factors. Anatomical classification is based on the level ofselectivity, for example, organ-specific, cell-specific, andorganelle-specific. Mechanistic targeting can be distinguished basedupon whether it is passive or active. Passive targeting utilizes thenatural tendency of liposomes to distribute to cells of thereticulo-endothelial system (RES) in organs which contain sinusoidalcapillaries. Active targeting, on the other hand, involves alteration ofthe liposome by coupling the liposome to a specific ligand such as amonoclonal antibody, sugar, glycolipid, or protein, or by changing thecomposition or size of the liposome in order to achieve targeting toorgans and cell types other than the naturally occurring sites oflocalization.

The surface of the targeted gene delivery system may be modified in avariety of ways. In the case of a liposomal targeted delivery system,lipid groups can be incorporated into the lipid bilayer of the liposomein order to maintain the targeting ligand in stable association with theliposomal bilayer. Various linking groups can be used for joining thelipid chains to the targeting ligand.

Fiducial Markers

Since the optogenetic reagents cannot be visualized within cells, it isuseful to have fiducial markers that can provide information on whetherendothelial cells received the recombinant nucleic acid moleculeencoding the optogenetic reagent and whether the optogenetic reagent maybe expressed in these cells. This is achieved by using fluorescentfiducial marker proteins well known in the art, e.g., green fluorescentprotein (GFP), red fluorescent protein (RFP), yellow fluorescent protein(YFP), and their natural and engineered variants. When expressed incells, and when contacted with the correct wavelength of light, theseproteins produce a fluorescent signal that can be visualized.

In one aspect of the invention, the fiducial marker is co-expressed withthe optogenetic reagent. This can be achieved by using two promoters inthe expression vector. One promoter drives the expression of theoptogenetic reagent, while the other promoter drives the expression ofthe fiducial marker protein. It is well accepted in the art that theexpression of the fiducial marker protein is an indication that theoptogenetic reagent is also expressed in the same cell. The twopromoters can be of the same kind or of different kinds. For example, aCMV promoter can drive the expression of the optogenetic reagent, whilean EF1-α promoter may drive expression of the fiducial marker.Alternatively both promoters can be CMV promoter.

In one aspect of the invention, the fiducial marker gene is cloned inthe same reading frame as the optogenetic element coding gene with aninternal ribosome entry site (IRES) in between the two open readingframes (ORFs). The IRES enables the co-translation of the fiducialmarker protein from the same mRNA that encodes the optogenetic reagent,thus producing two separate proteins. Alternatively, the IRES sequencecan be replaced by the 2A sequence known in the art. The 2A protein is aself cleaving peptide that when translated cleaves and releases theprotein translated downstream of it. Thus the fiducial marker protein istranslated and then released. In both of these methods the expression ofthe fiducial marker protein provides confirmation of the expression ofthe optogenetic reagent.

In another aspect of the invention, the fiducial marker gene is clonedas a fusion protein with the optogenetic reagent. Typically, a fiducialmarker protein is fused to the C-terminus (cytoplasmic side) of theoptogenetic reagent.

Light Sources and Delivery of Light for Regulating Permeability of CellsExpressing Optogenetic Reagents

Light Source

The permeability of ECs expressing the optogenetic reagents can bechanged by presentation, to these cells, of light of the appropriatewavelength. The source of the light can be one or more of a laser e.g.,a diode-pumped solid-state laser. Alternatively the light source can beone or more of a light emitting diode (LED), or an array of LEDsemitting light of a specific wavelength (Zorzos et al, Optics Letters,2010, 35:24, 4133; and in Bernstein et al, Proc. Soc. Photo Opt.Instrum. Eng., 2008, 6854:68540H, each of which is incorporated hereinby reference). In one aspect of the invention, the light source can bean array of multi-wavelength LEDs as described in Bernstein et al, 2008,for presenting light of different wavelengths. Additionally, shuttersand filters can be used to control the duration and wavelength of light.

Light Delivery

The invention features delivery of light using optical fibers. Thisapproach typically involves the use of a cannula that is implanted onthe area of the body, e.g., brain, where light will be presented. Thecannula allows for precise targeting of the optical fiber. The opticalfiber can be connected to a laser via a FC/PC connection. Typically theoptical fibers are placed using stereotactic guidance to deliver lightto a precise location.

In another aspect of the invention, light can be delivered through amulti-waveguide implantable probe that can deliver light of multiplewavelengths, e.g., blue and yellow light, to multiple target areas alongthe axis of the probe. The design of such a probe is described in detailin Zorzos et al, supra. Such a probe is useful for delivering light oftwo different wavelengths to precise areas of the vasculature and ECs todrive optogenetic reagents, e.g., step function opsins, thus providingprecise spatial and temporal control of when and where EC permeabilityis changed.

In yet another aspect of the invention, light can be delivered using animplantable prosthetic device containing 4D-LED arrays as described inBernstein et al, supra. Such a device does not use an expensive andbulky laser setup, and can be designed as a prosthetic device that canbe attached to a part of the body, e.g., the brain, for delivery oflight. In addition, 4D LED arrays allow the delivery of multiplewavelengths of light thus providing precise temporal and spatial controlof regulating the permeability of ECs.

In one aspect of the invention, light may be delivered using a lightdelivery catheter as described in U.S. Pat. No. 6,290,668, incorporatedherein by reference. Such catheters contain a guidewire and a lightguide and are approved for therapeutic uses, e.g., removal of bloodclots. With this approach, the BBB could be locally controlled at thesite of positioning of the fiber optic or fiber optics without surgicalimplantation or presentation of a fiber optic, making spatio-temporallylocalized control of the BBB relatively less invasive.

In another aspect of the invention, light may be provided by abioluminescent reaction. We and others (Berglund et al., PLoS One,8:e59759, 2013; incorporated herein by reference) have shown thatbioluminescent enzymes (commonly referred to by the name ‘luciferases’)can, in the presence of their required co-factor (small moleculescommonly referred to by the name ‘luciferins’), generate sufficientphoton production to drive robust optogenetic reactions. Luciferases canbe expressed in the same endothelial cell as the optogenetic elements orin other cells or vehicles to create the desired control of endothelialphysiology for the impact(s) described throughout the application.Luciferases dependent on other co-factors beyond luciferinpresentation—such as the well-described calcium dependence of theAequorin enzyme—can also be employed to allow activity-dependentregulation of endothelial physiology. Fusion proteins tetheringoptogenetic elements to bioluminescent enzymes (referred to as‘luminopsins’ in Berglund et al., 2013, supra) can also be employed, ascan many other combinations that place these two factors in sufficientproximity. Delivery of luciferins can occur through intravenousinjection, through direct infusion by a cannula, or through othermethods. These small molecules can in many instances cross theblood-brain barrier, removing a barrier to application.

The use of this specific mode of light production allows the control ofendothelial cells repeatedly through minimally invasive intravenousdelivery. For example, if the vasculature surrounding a tumor were madeto express luciferases and optogenetic elements, then at a later dateintravenous injection of an anti-tumor agent with a luciferinfacilitates delivery across the blood-brain barrier to enhance theconcentration of drug reaching the desired target. In anotherapplication, expression of such a combination of luciferase andoptogenetic elements allows for the continued induction ofvasoconstriction in the region of a tumor, potentially negativelyimpacting the tumor by reducing perfusion to it acutely, or throughimpairing angiogenesis.

Therapeutic Uses of the Invention

The invention features methods for controlling endothelial cells anduses of these methods for treating disorders where control of vascularproperties is involved. For example, control of EC permeability providesa method of controlling vascular permeability in the brain, e.g., bycontrolling the blood-brain barrier, (BBB), or in the rest of the body(e.g., by controlling the blood-lung barrier). This control may eitherbe exerted locally (e.g., in a single brain area) or globally (e.g.,throughout the brain or even the entire body). Control of ECpermeability may be used to facilitate delivery, from the blood to othertissues, of exogenous elements including drugs, cells, molecules,antibodies, and organic and inorganic compounds. Additionally, ECpermeability can be manipulated to block or decrease delivery of suchexogenous substances. Control of EC permeability may also be used tofacilitate the delivery from the blood to other tissues of endogenouselements such as hormones, nutrients, precursors in biochemicalprocesses (e.g., amino acids that will be converted to neurotransmittersin the central nervous system) and cells (e.g., immune cells).Additionally, EC permeability can be used to block or decrease deliveryof such endogenous elements. With tight optogenetic control over the BBBor other EC mediated barriers, opening and then closing it withprecision will allow higher drug concentration to be delivered to thepermeabilized area with lower IV infusion concentration (FIG. 1).Optogenetic vascular barrier opening could be achieved minimallyinvasively by catheterized fiber optics. Alternatively, intracranialfiber optic implantation is no more invasive than the approachescurrently used.

Therapeutic Uses in Treatment of Brain Disorders

The invention features methods that can facilitate the delivery oftherapeutics (e.g., drugs, small molecules, peptides, proteins,antibodies, nucleic acid molecules, and organic and inorganic compounds)to the brain, across the BBB, to treat brain disorders. For example, theBBB is a key challenge in neuro-oncology. Glioma prognosis (80% ofmalignant diagnoses) is poor. Glioblastoma multiforme, the most commonglioma, shows ˜18 month life expectancy.

Other diseases in which changes in BBB permeability could enhancedelivery of exogenous therapeutics include brain cancer, epilepsy,Parkinson's disease, Alzheimer's disease, neurological diseases of othertypes, and psychiatric diseases (e.g., by selective delivery ofanti-depressants to relevant brain regions and not other unintendedtargets).

In case of several diseases, control of BBB permeability to endogenousfactors could improve disease outcomes. For example, inappropriatepermissiveness of the BBB is a major source of several brain diseasesincluding Alzheimer's Disease (AD), Multiple Sclerosis (MS), malaria,and meningitis, all of which show inappropriate transmission across theBBB. In AD, amyloid-3 accumulation in the brain is believed to resultfrom increased influx across EC and/or decreased efflux after brainaccumulation. Enhancing the BBB could be beneficial in AD, and in caseof the present invention—which selectively targets EC and can be made tospan the entire vasculature non-invasively—is advantageous. The targetedvariants of the present invention could also be useful in MS, whichshows a localized failure in barrier properties and is treated byblockade of leukocyte permeability. Increased blockade at the site oflesion can be increased and could potentially enhance the BBB only atthat position, allowing its normal function in the rest of the BBB.

Therapeutic Uses in Control of Vascular Tone and Arterial Diameter

Control of ECs can be used to control vascular tone and directly linkedphenomena such as blood flow rate, blood volume, and blood pressure,including direct induction of vasoconstriction or vasodilation. In oneaspect of the invention, the method provides bi-directional control ofvessel resistivity to manipulation by other sources and can help inprevention or decrease of constriction or dilation by endogenous factorsin the body (e.g., that occur as a symptom of a disease), or byexogenous factors administered (e.g., blocking the vascular impact of adrug that has a different therapeutic target).

In another aspect of the invention, the method provides a way for theenhancement of constriction or dilation by endogenous factors in thebody (e.g., promoting metabolism-driven dilation/hyperemia) or exogenousfactors administered (e.g., potentiating the efficacy of blood-pressuremedication). Several medical conditions and/or diseases enhance ordiminish the natural capacity to control blood flow, volume and/orpressure, or the ability to maintain vascular tone. In the extreme,damage in most strokes and aneurysms may be considered such problems,and a host of other diseases including diabetes, hypertension, varicoseveins, erectile dysfunction, and variants of cardiac disease fit thisdescription. As such, the ability to control vascular tone is a majortherapeutic target. Further, the delivery of blood-borne factors (e.g.,exogenous or endogenous agents) could also be enhanced or retarded byincreased blood flow and/or volume to a region, thereby enhancing drugdelivery and/or nutrient supply. Also, this application of vascularcontrol could be introduced for acute treatment contexts, such asanticipated need for reducing blood flow to a region during surgery. Inthis application, patients would be pre-treated with manipulations thatachieve EC optogenetic expression, so that light could be used in theprocedure. Another such relevant application would be in advance ofindividuals anticipating potential injury (e.g., soldiers), whereachieving control over vascular tone with light could allow moreefficient treatment in potentially adverse contexts (e.g., abattlefield).

Therapeutic Uses in Control of Vascular Growth

In yet another aspect of the invention, optogenetic control of ECsprovides a means of manipulating ECs to control vascular growth andretraction in any part of the body. Several diseases, including stroke,cerebrovascular epilepsies, retinopathies (e.g., macular degeneration)and cancer, are typified by maladaptive growth of vasculature(angiogenesis). Conversely, several diseases are characterized byinsufficient ingrowth e.g., into ischemic tissue zones. Perturbation ofEC is known to promote or retard such vascular growth depending on thenature of the stimulation. As such, regulation of EC physiology bydirect and likely repeated activation or inactivation throughoptogenetic means could provide a direct means for retarding orstimulating vascular elaboration.

Therapeutic Uses in Treatment of Lung Cancer

The methods described above, for the brain, will provide generalinfrastructure/methods for innovations in delivery across, orreinforcement of, the blood-lung barrier. As one example, optogeneticregulation of EC membrane properties in the lung could improve efficacyand targeting of adjuvant chemotherapy to improve drug penetration innon-small cell lung carcinoma.

EXAMPLES Example 1 Modulation of EC Permeability in the BBB UsingHalorhodopsin-3.0

Halorhodopsin-3.0 was selectively expressed in mouse ECs using IV viralinjection. After ˜6 weeks, 2-photon imaging of the in vivo neocortex (astill image is shown in FIG. 2A) was used to test the impact ofoptogenetic stimulation on the BBB. To measure extravasation,rhodamine-dextran (10 kD), which does not typically cross the BBB, wasinjected via IV. Amber light pulses (optimized for Halorhodopsin) werepresented for 10 seconds. Following stimulation, a discrete increase influorescence in neocortex surrounding the vessel occurred (ROIs 1 and3/time series) with a decrease in intra-arterial emission (ROI2),indicating movement of rhodamine-dextran across the BBB into the brain(Please see FIG. 2A and FIG. 2B). Vessel walls were stationarythroughout, and control experiments in non-transduced mice did not showthese effects. This BBB opening was discrete in time, <15 seconds.

Example 2 Second Example of Modulation of EC Permeability in the BBBUsing Halorhodopsin-3.0

Halorhodopsin-3.0 was selectively expressed in mouse ECs usingtransgenic mice bred to encode Cre-recombinase in ECs, and LoxPsequences flanking the fusion protein Halorhodopsin (eNpHR 3.0)-EnhancedYellow Fluorescent Protein (Halo3.0-EYFP).

After ˜6 weeks, 2-photon imaging of the in vivo neocortex (a still imageis shown in FIG. 3A) was used to test the impact of optogeneticstimulation on the BBB. To measure extravasation, rhodamine-dextran (10kD), which does not typically cross the BBB, was injected via IV. Amberlight pulses (optimized for Halorhodopsin) were presented for 10seconds. Following stimulation (three independent presentations of thelight), a discrete increase in fluorescence in neocortex surrounding thevessel occurred (ROIs 1/time series) with a small decrease inintra-arterial emission on trials 2 and 3 (ROI 2), indicating movementof rhodamine-dextran across the BBB into the brain (FIG. 3A and FIG.3B).

Example 3 Naturally-Occurring Changes in Vascular Width

The fusion protein Channelrhodopsin-2-Enhanced Yellow FluorescentProtein (Ch2R-EYFP) was selectively expressed in mouse ECs usingtransgenic mice bred to encode Cre-recombinase in ECs, and LoxPsequences flanking the fusion protein Channelrhodopsin-2-Enhanced YellowFluorescent Protein (Ch2R-EYFP).

Observation of fluorescence using 2-photon microscopy confirmed theexpression of Ch2R-EYFP in endothelial cells of a mouse arteriolelocalized above the primary sensory neocortical representation of thevibrissae (“whiskers”) on the face of the mouse (FIG. 4A and FIG. 4B).The mouse vibrissae were stimulated by brief deflections applied at 5 Hzfor 10 seconds. As is typical in our data, within hundreds ofmilliseconds of vibrissal deflection, a vascular width change isobserved (FIG. 4A and FIG. 4C). In this case, the width change was adilation that sustained for ˜20 seconds. As shown in FIG. 4D, vascularconstriction can be another observed vascular response to sensory drive.Not pictured are examples that are commonly observed in which naturaldilations or constrictions are followed by large ‘rebound’ or‘overshoot’ width changes in the opposite direction to that induced withsensory drive.

Example 4 Control of Vascular Dilation with Endothelial OptogeneticDrive

Halorhodopsin-3.0 was selectively expressed in mouse ECs usingtransgenic mice bred to encode Cre-recombinase in ECs, and LoxPsequences flanking the fusion protein Halorhodopsin (eNpHR 3.0)-EnhancedYellow Fluorescent Protein (Halo3.0-EYFP). An arteriole localized withinthe neocortex of a mouse (exemplary arteriole expressing Halo3.0-EYFP isshown in FIG. 5A). Amber light (560 nm) was administered as repeatedbrief pulses for the period indicated by the amber background in FIG. 5Bto an arteriole in the neocortex of an awake mouse to elicit vasculardilation (FIG. 5B). As with many naturally-occurring events, theseinduced effects are sometimes followed (as shown here) by overshoot inthe opposite direction.

Example 5 Control of Vascular Constriction with Endothelial OptogeneticDrive

The fusion protein Channelrhodopsin-2-Enhanced Yellow FluorescentProtein (Ch2R-EYFP) was selectively expressed in mouse ECs usingtransgenic mice bred to encode Cre-recombinase in ECs, and LoxPsequences flanking the fusion protein Channelrhodopsin-2-Enhanced YellowFluorescent Protein (Ch2R-EYFP). An arteriole localized within theneocortex of a mouse (exemplary arteriole expressing Ch2R-EYFP is shownin FIG. 5A). Blue light (470 nm) was administered for the periodindicated by the blue background in FIG. 5C to an arteriole in theneocortex of an awake mouse to elicit vascular constriction (FIG. 5C).As with many naturally-occurring events, these induced effects aresometimes followed (as shown here) by overshoot in the oppositedirection.

Other Embodiments

While certain novel features of this invention shown and described arepointed out in the annexed claims, the invention is not intended to belimited to the details specified, since a person of ordinary skill inthe relevant art will understand that various omissions, modifications,substitutions and changes in the forms and details of the inventionillustrated and in its operation may be made without departing in anyway from the spirit of the present invention. No feature of theinvention is critical or essential unless it is expressly stated asbeing “critical” or “essential.”

Those skilled in the art will recognize or be able to ascertain using nomore than routine experimentation many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed in the scope of the present invention.

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each independent publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

What is claimed is: 1-2. (canceled)
 3. A method to deliver a therapeuticacross the vascular endothelial barrier comprising endothelial cellsexpressing an optogenetic reagent in a subject, said method comprisingthe steps of: a) introducing said therapeutic into the blood stream ofsaid subject; and b) contacting said endothelial cells with light,wherein said light activates said optogenetic reagent and therebychanges permeability of said endothelial cells and opens the vascularendothelial barrier such that said therapeutic in the blood streamcrosses the vascular endothelial barrier.
 4. The method of claim 3,wherein said optogenetic reagent is selected from the group consistingof ChR1, ChR2, VChR1, ChR2 C128A, ChR2 C128S, ChR2 C128T, ChD, ChEF,ChF, ChIEF, NpHR, eNpHR, Arch 3.0, Arch T 3.0, Mac 3.0, melanopsin,chimeras of these proteins and natural and engineered variants thereof.5. The method of claim 3, wherein said optogenetic reagent are expressedin the endothelial cells by introducing a recombinant nucleic acidencoding the optogenetic reagent into said cells or precursors thereof.6. The method of claim 5, wherein said recombinant nucleic acid isintroduced into said cells by using any one or more of a virus, anelectroporation device, a transfection method, and a transgenic method.7. The method of claim 6, wherein said recombinant nucleic acid isencapsidated within a recombinant virus selected from the groupconsisting of recombinant adeno-associated virus (AAV), recombinantretrovirus, recombinant lentivirus, recombinant poxvirus, recombinantrabies virus, recombinant pseudo-rabies virus, and recombinant herpessimplex virus, and human immunodeficiency virus (HIV).
 8. The method ofclaim 7, wherein said recombinant nucleic acid encoding said optogeneticreagent further encodes a fiducial marker, that when expressedidentifies cells infected by the recombinant virus.
 9. The method ofclaim 8, wherein said fiducial marker is a fluorescent protein.
 10. Themethod of claim 6, wherein said virus is applied using intra-venousinjection or applied locally to infect endothelial cells in specificregions.
 11. The method of claim 3, wherein said light is applied usinga laser or a light emitting diode, and wherein application of light isrestricted to a defined spatial region of the body.
 12. The method ofclaim 11, wherein said light is delivered using a fiber optic cable orcatheter. 13-29. (canceled)
 30. The method of claim 3, wherein saidendothelial cells are part of a blood-air barrier in the lung.
 31. Themethod of claim 30, wherein said method is used to treat a disease ofthe lung.
 32. The method of claim 31, wherein said disease is a lungcancer.
 33. The method of claim 3, wherein said therapeutic is selectedfrom the group consisting of drugs, small molecules, peptides, proteins,antibodies, and nucleic acid molecules.